Project Summary: Human pluripotent stem cells provide an attractive experimental platform for studying the normal and abnormal development of human neural networks. In vitro, induced pluripotent stem cells (iPSC) have been used to study early developmental mechanisms and the initial stages of synaptogenesis and circuit formation. iPSC also offer the tantalizing but unrealized potential to study human neural circuits in vitro. Our efforts to model the development of human cortical circuits in vitro indicate that differentiation can be segmented into a 5-step progression involving: 1) patterning of the neuroectoderm and early neurogenesis - robustly evident after 3 weeks; 2) neuroblast migration and initial differentiation into regionally-specific neuronal subtypes - evident after 6 weeks; 3) development of spontaneous electrical activity and early synaptogenesis ? evident after 2-3 months; 4) initial appearance of astrocytes and the formation of vigorous and synchronous oscillatory bursting within large ensembles ? evident within 3-5 months; 5) resolution of large hypersynchronous neuronal ensembles into smaller, discreetly firing sub-ensembles ? occasionally observed after more than 6 months. Appearance of oligodendrocyte progenitors and myelination of axons seen in vivo has not yet been observed or reported. Although it is comforting to confirm that human-specific developmental programs are temporally intact in iPSC models, the protracted timeline makes it challenging to study late developmental mechanisms or mature circuit function. The ultimate goal of this project is to develop methods to accelerate the formation of networks of highly interconnected human neurons with mature synapses. Synchronized and oscillatory neuronal activity appears to be a fundamental and obligate transitional property of developing nervous systems. Young neurons in structures as diverse as retina, cerebral cortex and spinal cord experience periods of high connectivity and robust neuronal activity that are subsequently refined to produce specific synaptic connections and regulated action potential activity. Human neurons developing in vitro presumably require the acquisition of these same properties in order to become functional neuronal networks. Our preliminary work suggests that these obligate properties are acquired by the interactions of cell types generated at different times in development and/or from different regions of the developing brain. Experiments in this proposal focus on novel methods that recapitulate these interactions to accelerate the acquisition of synchronous oscillatory bursting and promote the further maturation of iPSC-derived human neural networks.